BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
48,
137-142 (1992)
Glycoprotein Metabolism in Neuronal Ceroid Lipofuscinosis Fibroblasts J. HEANEY-KIERAS,*‘$
F. J. KIERAS,* AND K. E. WISNIEWSKI~
Departments of *Human Genetics and tPathologica1 Neurobiology, New York State Ofice of Mental Retardation and Developmental Disabilities, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314 Received May 11, 1992
Human skin fibroblast lines of the infantile form of neuronal ceroid lipofuscinosis and control lines were cultured in the presence of [‘Hlglucosamine plus [3H]mannose and [?S]methionine. The labeled glycoconjugates were compared by quantitative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The infantile form of the disease showed a 75% decrease of four glycoprotein components of M, 120-140 kDa. These components appeared to be N-linked glycoproteins as peptide-N’-( N-acetyl-P-glucosaminyl) asparagine amidase (PNGase F) released 86-96% of the labeled carbohydrate from the labeled protein. These results suggest that the infantile form of this disease may be characterized by abnormalities in glycoconjugate metabolism leading to reduction of specific glycoproteins. Q 1992 Academic
Press. Inc.
Neuronal ceroid lipofuscinoses (NCL)’ are among the most common progressive brain diseases of childhood in western countries with an incidence estimated at 1 in 25,000 live births (1). NCL are named for the accumulation of autofluorescent pigments (ceroid and lipofuscin) in neural and extraneural tissues. The three childhood forms of NCL have been clinically categorized by age of onset and the course of deterioration. The infantile form is the most severe and has an age of onset about 6 months (2). The biochemical defect in NCL is not known for any form of NCL, but various investigators have suggested defects in oxidation of polyunsaturated fatty acids (3), in the metabolism of dolichol linked oligosaccharides (4,5), in the lysosomal degradation of proteins (6,7), and in the metabolism of N-and O-linked oligosaccharides of glycoproteins (2,8-13). ’ Abbreviations used: NCL, neuronal ceroid lipofuscinoses; PAGE, polyacrylamide gel electrophoresis; PNGase F, peptide-p-(N-acetyl$-glucosaminyl) asparagine amidase; SDS, sodium dodecyl sulfate. 137 08854505192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved
138
HEANEY-KIERAS,
KIERAS,
AND WISNIEWSKI
METHODS AND MATERIALS
NCL cell lines from the Institute for Basic Research cell bank and normal controls of ages 14 months to adult from the Human Genetic Mutant Cell Repository (NJ) were used. Fibroblast lines in monolayer culture were grown in 20 &i/ml each [3H]glucosamine and [3H]mannose and 28.5 &i/ml [35S]methionine (American Radiolabelled Chemicals, Inc., MO, and Amersham Radiochemicals, MA) in fetal calf serum (12%) and RPM1 1640 containing medium for 66 h. Washed monolayers were lysed in 0.5% SDS in phosphate-buffered saline, pH 7.4, with protease inhibitors (phenylmethylsulfonylfloride, 6-aminohexanoate, and benzamidine (Sigma Chemicals, Inc., St. Louis, MO)), heated for 10 min at 100°C and clarified by centrifugation (lO,OOOg, 20 min). Acid precipitable radioactivity was determined in 10% cold trichloroacetic acid. Protein was determined as described by Lowry et al. (14). Molecular weight standards were purchased (BioRad Laboratories, CA, and Diversified Biotech, MA). Equal amounts of radioactivity for each sample were placed on each gel. SDS-PAGE (7.5% acrylamide) was performed according to Laemmli (15) on 1.5 or 3.0 x 120 mm slab gels; after overnight fixation (30% methanol, 7% acetic acid) lanes were cut into lmm slices, the acrylamide was depolymerized in H202, scintillant (Ecolume, NEN, MA) was added, and the radioactivity was determined in a Packard Tricarb Counter. Labeled material was extracted from macerated gel slices in buffer for PNGase F, an enzyme with specificity for all classes of N-linked oligosaccharides (16) (N-Glycanase, Genzyme Corp., MA) and digested according to Hirani et al. (17). The digest was adjusted to 90% cold acetone (18) and the radioactivity released by digestion was separated by centrifugation (Beckman Microfuge 12) or the digest was applied to a Sephadex G-50 column as described (2,18). In labeled fibroblast lines of the juvenile form of NCL we have shown that most (>90%) of the radioactivity ([3H]glucosamine and [3H]mannose) may be released from glycoproteins by PNGase F treatment (2). RESULTS
Skin fibroblast glycoconjugates containing carbohydrate and protein labels were analyzed by SDS-PAGE. Under conditions which favor separation of higher molecular weight components, the region of M, 120-140 kDa showed about 25% of the radioactivity in the infantile form of NCL compared to control (Fig. 1). The component(s) of M, 200 kDa appeared to be labeled equally in affected and control lines and has been used as an “internal” marker. We have examined two additional NCL infantile form cell lines and three control cell lines and have observed similar profiles to those presented here (results not shown). These components were then isolated from gels after SDS-PAGE and divided into four peaks of radioactivity between M, 120 and 140 kDa plus the M, 200 kDa internal marker. In Table 1 the results for two of the infantile form lines of NCL and two control fibroblast lines are given. The levels of radioactivity in the M, 200 kDa components are similar in affected and control lines. The amount of radioactivity in the carbohydrate portion of glycoconjugates in the M, 120-140 kDa region is about 5% of the total acid precipitable radioactivity in a lysate. In
GLYCOPROTEIN
t
METABOLISM
Relative
Origin
139
IN NCL
Distance
FIG. 1. Quantitative SDS-PAGE analysis (7.5% acrylamide) of radiolabeled glycoproteins of human skin fibroblasts in control (C), (upper) and infantile form of NCL (INCL), (lower) lines. Radioactivity in 1 mm slices of gel. Location of protein standards indicated by arrows (upper). Equal amounts of radioactivity were placed on each gel.
Quantitative SDS-PAGE
TABLE 1 of Radiolabeled Peaks M, 120-140 kDa in Infantile Form of NCL and Normal Control Fibroblasts Radioactivity (cpm) peaks between M, 120 and 140 kDa
M, 200 Cell line” Cl c2 INCLl INCL2
Isotope
kDab
1
2
3
4
‘H ‘3 )H “S 3H 35S 3H ?3
1799(87) 3760(11) 1613(91) 3792( 6) 1874(90) 3898( 8) 1711(92) 3358( 4)
946(88) 1820( 9) 944(90) 1532(11) 249(86) 499( 8) 213(92) 487( 11)
1328(87) 3120( 15) 1112(90) 2502( 9) 332(91) 670( 3) 379( 87) 498( 7)
2284(92) 5130(11) 2617(94) 4424( 6) 565(90) 796( 8) 332(88) 611(10)
2951(90) 5970( 7) 3029(96) 5356( 6) 734(92) 1660( 10) 662(91) 780( 7)
’ Cell lines are designated C for control and INCL for infantile form of NCL. b Equal amounts of radioactivity are applied to each gel. Radioactivity in peak is coincident with M, 200kDa standard protein found in both C and INCL cells and used as an internal standard. ’ Percentage of radioactivity that is sensitive to PNGase F is noted in parentheses.
140
HEANEY-KIERAS,
KIERAS,
AND WISNIEWSKI
addition, the radioactivity in the carbohydrate portions of these components was susceptible to PNGase F, consistent with an N-linked type of oligosaccharide, as may be seen by the numbers in parentheses in Table 1. The separation of the carbohydrate from the protein radioactivity may be accomplished by precipitation in 90% acetone as shown here or by separation of the protein label from the carbohydrate label by Sephadex G-50 column chromatography (2) (results not shown). To compare the infantile form with the juvenile form of NCL (results not shown), we labeled glycoconjugates in the same manner and separated the glycoproteins by SDS-PAGE. In two fibroblast lines of the juvenile form of NCL, the M, 120-140 kDa region had radioactivity in protein and carbohydrate similar in amounts to control lines. Furthermore, the M, 120-140 kDa material was sensitive (over 85% carbohydrate label released) by PNGase F consistent with glycoproteins containing N-linked oligosaccharides. DISCUSSION Abnormalities in glycoconjugate metabolism have been reported to be due to incorrect carbohydrate transfer and processing, improper protein structure, or lack of gene expression (see review (19)). In NCL, abnormalities in glycoconjugate metabolism particularly in the carbohydrate portion have been reported in the brain (9,11-13) skin (lo), and cultured fibroblasts (2,8) and the defects usually involve the high mannose type of N-linked oligosaccharide. In studies of the accumulated oligosaccharyldiphosphodolichols of NCL, the oligosaccharides appear to be metabolites of the normal glycosylation intermediate Glc3MangGlcNA@ (20-22), w h ic h would result from a block in the transfer to a protein acceptor or a deficiency in processing enzymes. In this study both carbohydrate and protein of the M, 120-140 kDa glycoproteins were diminished and either or both may be defective. Since there are several reports of the carbohydrate defects in NCL, two instances of other disorders may be cited in which the incorrect structure of N-linked carbohydrate leads to degradation of the glycoproteins. In a disorder of sucrase-isomaltase, an enzyme of the intestinal brush border, the high mannose precursor is transferred properly to the protein, but processing to the mature complex oligosaccharide is defective leading to degradation of the glycoprotein (23). In the familial form of hypercholesterolemia, one defect of the LDL receptor is an incorrect processing of the precursor oligosaccharide leading to degradation of the glycoprotein (24). The three childhood forms of NCL are classified clinically by age of onset and course of the disease and, although the forms share characteristics of vision loss, seizures, myoclonus, and dementia, it is not clear whether these are separate diseases since few biochemical studies have compared the childhood forms. The juvenile and infantile forms have autosomal inheritance. Recent genetic work has assigned the infantile form in Finnish families to Chromosome 1 (25) and the juvenile form in western European families to Chromosome 16 (26). In a study of oligosaccharides derived from dolichol intermediates, the juvenile form had smaller average size and the infantile form larger size (20-22). In the present study of labeled glycoconjugates of fibroblasts, the infantile form showed a 75%
GLYCOPROTEIN
METABOLISM
IN NCL
141
reduction of M, 120-140 kDa glycoproteins while the juvenile form did not, suggesting that the two forms of NCL differ qualitatively at least in these components. These differences may be useful in the diagnosis of these two forms of NCL. ACKNOWLEDGMENTS This work was supported in part by funds from NIH Grant NS 23717, BRSG 2-SO7 RR05838-11, and by funds from the NYS Office of Mental Retardation and Developmental Disabilities.
REFERENCES 1. Zeman W. The neuronal ceroid-lipofuscinoses. Prog NeuropathoZ3:203-223, 1976. 2. Wisniewski KE, Rapin I, Heaney-Kieras J. Clinico-pathological variability in the childhood neuronal ceroid-lipofuscinoses and new observations on glycoprotein abnormalities. Am J Med Genet Suppl5:27-46, 1988. 3. Siakotos AN, Bray R, Dratz E, Van Kuijk F, Sevanian A, Koppang N. 4-Hydroxynonenal: A specific indicator for canine neuronal-retinal ceroidosis. Am J Med Genet Suppl5:171-181, 1988. 4. Pullarkat RK, Kim KS, Sklower SL, Pate1 VK. Ohgosaccharyl diphosphodolichols in the ceroidhpofuscinoses. Am J Med Genet Suppl5:243-251, 1988. 5. Hall NA, Patrick AD. Accumulation of dolichol-linked oligsaccharides in ceroid-lipofuscinosis (Batten Disease). Am J Med Genet Suppl5:221-232, 1988. 6. Palmer DN, Martinus RD, Barns G, Reeves RD, Jolly RD. Ovine ceroid-lipofuscinosis 1:Lipopigment composition is indicative of a lysosomal proteinosis. Am J Med Genet SuppZ5:141158, 1988. 7. Dawson G, Glaser PT. Abnormal Cathepsin B activity in Batten Disease. Am J Med Genet Suppl 5:209-220, 8.
9. 10. 11.
12. 13. 14. 15. 16.
1988.
Dawson G. Approaches to the detection of neuronal ceroid lipofuscinosis in cultured skin tibroblasts. In Ceroid-Lipofuscinosis (Batten’s Disease) (Amrstrong D, Koppang N, Rider JA, Eds.). Amsterdam: Elsevier, 1982, pp 229-240. Krusius T, Viitala J, Palo J, Maury CP. Enrichment of high mannose-type glycans in nervous tissue glycoproteins in neuronal ceroid-lipofuscinosis. J Neural Sci 72:1-10, 1986. Wisniewski KE, Szumanska G. The ultrastructural observation and histochemical localization of some glycoconjugates in neuronal ceroid-lipofuscinosis. 10th Intl Congr Neuropathol, Stockholm, Sweden (No.799.2) p 396, 1986. Wisniewski KE, Maslinska D, Kitaguchi T. High level mannose type of glycopeptides in brains of human neuronal ceroid-lipofuscinosis (HNCL) and canine experimental model (CNCL). J Neuropath Exp Neural 47~327, 1988. Elleder M. Lectin histochemical study of lipopigments with special regard to neuronal ceroidlipofuscinosis. Results with concanavalin A. Histochemistry 93197-205, 1989. Wisniewski KE, Maslinska D. Lectin histochemistry in brains with juvenile form of neuronal ceroid-lipofuscinosis (Batten Disease). Acta Neuropathol80:274-79, 1990. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the fohn-phenol reagent. J Biol Chem 193~265-275, 1951. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685, 1970. Tarentino AL, Plummer TH Jr. Peptide-N“-(N-acetyl-P-glucosaminyl) asparagine amidase and endo-/3-N-acetylglucosaminidase from Flavobacterium meningosepticum. Methods Enzymol D&770-778,
1987.
Hirani S, Bernasconi RJ, Rasmussen JR. Use of N-Glycanase to release asparagine-linked oligosaccharides for structural analysis. Anal Biochem 162~485-492, 1987. 18. Freeze HH, Varki A. Endo-glycosidase F and peptide N- glycosidase F release the great majority of total cellular N-linked oligosaccharides: Use in demonstrating that sulfated N-linked oligosaccharides are frequently found in cultured cells. Biochem Siophys Res Commun 140~967-973, 1986. 17.
142
HEANEY-KIERAS,
KIERAS,
AND WISNIEWSKI
19. Rademacher TW, Parekh RB, Dwek RA. Glycobiology. Annu Rev Biochem 57~785-838, 1988. 20. Hall NA, Patrick AD. Analysis of dolichol-linked oligosaccharides in brains from patients with Batten’s disease. B&hem Sot Truns 16:1031-1032, 1988. 21. Hall NA, Patrick AD. Identification of intact dolichol-linked oligosaccharides in the brains of patients with ceroid lipofuscinosis (Batten’s disease). J Inher Metub Dis Suppl2:379-382, 1989. 22. Hall NA, Haltia M, Patrick AD. High mannose dolichol-linked oligosaccharides in infantile ceroid lipofuscinosis. Biochem Sot Trans 17~1032-1033, 1989. 23. Lloyd ML, Olsen WA. A study of the molecular pathology of sucrase-isomaltase deficiency. N Engl J Med 316~438-442, 1987. 24. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science 23234-47, 1986. 25. Jarvell I, Mbnpil J, Haataja L, Santovouri P, Aula P, Peltonen L. Assignment of infantile form of neuronal ceroid-lipofuscinosis (INCL) to the short arm of chromosome 1. Am J Med Genet 47A185, 1990. 26. Eiberg H, Gardiner RM, Mohr J. Batten disease (Spielmeyer-Sjogren disease) and haptoglobins (HP): Indication of linkage and assignment to chr. 16. Clin Genet 36~217-218, 1989.
MEDICINE
AND
METABOLIC
BIOLOGY
48,
137-142 (1992)
Glycoprotein Metabolism in Neuronal Ceroid Lipofuscinosis Fibroblasts J. HEANEY-KIERAS,*‘$
F. J. KIERAS,* AND K. E. WISNIEWSKI~
Departments of *Human Genetics and tPathologica1 Neurobiology, New York State Ofice of Mental Retardation and Developmental Disabilities, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314 Received May 11, 1992
Human skin fibroblast lines of the infantile form of neuronal ceroid lipofuscinosis and control lines were cultured in the presence of [‘Hlglucosamine plus [3H]mannose and [?S]methionine. The labeled glycoconjugates were compared by quantitative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The infantile form of the disease showed a 75% decrease of four glycoprotein components of M, 120-140 kDa. These components appeared to be N-linked glycoproteins as peptide-N’-( N-acetyl-P-glucosaminyl) asparagine amidase (PNGase F) released 86-96% of the labeled carbohydrate from the labeled protein. These results suggest that the infantile form of this disease may be characterized by abnormalities in glycoconjugate metabolism leading to reduction of specific glycoproteins. Q 1992 Academic
Press. Inc.
Neuronal ceroid lipofuscinoses (NCL)’ are among the most common progressive brain diseases of childhood in western countries with an incidence estimated at 1 in 25,000 live births (1). NCL are named for the accumulation of autofluorescent pigments (ceroid and lipofuscin) in neural and extraneural tissues. The three childhood forms of NCL have been clinically categorized by age of onset and the course of deterioration. The infantile form is the most severe and has an age of onset about 6 months (2). The biochemical defect in NCL is not known for any form of NCL, but various investigators have suggested defects in oxidation of polyunsaturated fatty acids (3), in the metabolism of dolichol linked oligosaccharides (4,5), in the lysosomal degradation of proteins (6,7), and in the metabolism of N-and O-linked oligosaccharides of glycoproteins (2,8-13). ’ Abbreviations used: NCL, neuronal ceroid lipofuscinoses; PAGE, polyacrylamide gel electrophoresis; PNGase F, peptide-p-(N-acetyl$-glucosaminyl) asparagine amidase; SDS, sodium dodecyl sulfate. 137 08854505192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved
138
HEANEY-KIERAS,
KIERAS,
AND WISNIEWSKI
METHODS AND MATERIALS
NCL cell lines from the Institute for Basic Research cell bank and normal controls of ages 14 months to adult from the Human Genetic Mutant Cell Repository (NJ) were used. Fibroblast lines in monolayer culture were grown in 20 &i/ml each [3H]glucosamine and [3H]mannose and 28.5 &i/ml [35S]methionine (American Radiolabelled Chemicals, Inc., MO, and Amersham Radiochemicals, MA) in fetal calf serum (12%) and RPM1 1640 containing medium for 66 h. Washed monolayers were lysed in 0.5% SDS in phosphate-buffered saline, pH 7.4, with protease inhibitors (phenylmethylsulfonylfloride, 6-aminohexanoate, and benzamidine (Sigma Chemicals, Inc., St. Louis, MO)), heated for 10 min at 100°C and clarified by centrifugation (lO,OOOg, 20 min). Acid precipitable radioactivity was determined in 10% cold trichloroacetic acid. Protein was determined as described by Lowry et al. (14). Molecular weight standards were purchased (BioRad Laboratories, CA, and Diversified Biotech, MA). Equal amounts of radioactivity for each sample were placed on each gel. SDS-PAGE (7.5% acrylamide) was performed according to Laemmli (15) on 1.5 or 3.0 x 120 mm slab gels; after overnight fixation (30% methanol, 7% acetic acid) lanes were cut into lmm slices, the acrylamide was depolymerized in H202, scintillant (Ecolume, NEN, MA) was added, and the radioactivity was determined in a Packard Tricarb Counter. Labeled material was extracted from macerated gel slices in buffer for PNGase F, an enzyme with specificity for all classes of N-linked oligosaccharides (16) (N-Glycanase, Genzyme Corp., MA) and digested according to Hirani et al. (17). The digest was adjusted to 90% cold acetone (18) and the radioactivity released by digestion was separated by centrifugation (Beckman Microfuge 12) or the digest was applied to a Sephadex G-50 column as described (2,18). In labeled fibroblast lines of the juvenile form of NCL we have shown that most (>90%) of the radioactivity ([3H]glucosamine and [3H]mannose) may be released from glycoproteins by PNGase F treatment (2). RESULTS
Skin fibroblast glycoconjugates containing carbohydrate and protein labels were analyzed by SDS-PAGE. Under conditions which favor separation of higher molecular weight components, the region of M, 120-140 kDa showed about 25% of the radioactivity in the infantile form of NCL compared to control (Fig. 1). The component(s) of M, 200 kDa appeared to be labeled equally in affected and control lines and has been used as an “internal” marker. We have examined two additional NCL infantile form cell lines and three control cell lines and have observed similar profiles to those presented here (results not shown). These components were then isolated from gels after SDS-PAGE and divided into four peaks of radioactivity between M, 120 and 140 kDa plus the M, 200 kDa internal marker. In Table 1 the results for two of the infantile form lines of NCL and two control fibroblast lines are given. The levels of radioactivity in the M, 200 kDa components are similar in affected and control lines. The amount of radioactivity in the carbohydrate portion of glycoconjugates in the M, 120-140 kDa region is about 5% of the total acid precipitable radioactivity in a lysate. In
GLYCOPROTEIN
t
METABOLISM
Relative
Origin
139
IN NCL
Distance
FIG. 1. Quantitative SDS-PAGE analysis (7.5% acrylamide) of radiolabeled glycoproteins of human skin fibroblasts in control (C), (upper) and infantile form of NCL (INCL), (lower) lines. Radioactivity in 1 mm slices of gel. Location of protein standards indicated by arrows (upper). Equal amounts of radioactivity were placed on each gel.
Quantitative SDS-PAGE
TABLE 1 of Radiolabeled Peaks M, 120-140 kDa in Infantile Form of NCL and Normal Control Fibroblasts Radioactivity (cpm) peaks between M, 120 and 140 kDa
M, 200 Cell line” Cl c2 INCLl INCL2
Isotope
kDab
1
2
3
4
‘H ‘3 )H “S 3H 35S 3H ?3
1799(87) 3760(11) 1613(91) 3792( 6) 1874(90) 3898( 8) 1711(92) 3358( 4)
946(88) 1820( 9) 944(90) 1532(11) 249(86) 499( 8) 213(92) 487( 11)
1328(87) 3120( 15) 1112(90) 2502( 9) 332(91) 670( 3) 379( 87) 498( 7)
2284(92) 5130(11) 2617(94) 4424( 6) 565(90) 796( 8) 332(88) 611(10)
2951(90) 5970( 7) 3029(96) 5356( 6) 734(92) 1660( 10) 662(91) 780( 7)
’ Cell lines are designated C for control and INCL for infantile form of NCL. b Equal amounts of radioactivity are applied to each gel. Radioactivity in peak is coincident with M, 200kDa standard protein found in both C and INCL cells and used as an internal standard. ’ Percentage of radioactivity that is sensitive to PNGase F is noted in parentheses.
140
HEANEY-KIERAS,
KIERAS,
AND WISNIEWSKI
addition, the radioactivity in the carbohydrate portions of these components was susceptible to PNGase F, consistent with an N-linked type of oligosaccharide, as may be seen by the numbers in parentheses in Table 1. The separation of the carbohydrate from the protein radioactivity may be accomplished by precipitation in 90% acetone as shown here or by separation of the protein label from the carbohydrate label by Sephadex G-50 column chromatography (2) (results not shown). To compare the infantile form with the juvenile form of NCL (results not shown), we labeled glycoconjugates in the same manner and separated the glycoproteins by SDS-PAGE. In two fibroblast lines of the juvenile form of NCL, the M, 120-140 kDa region had radioactivity in protein and carbohydrate similar in amounts to control lines. Furthermore, the M, 120-140 kDa material was sensitive (over 85% carbohydrate label released) by PNGase F consistent with glycoproteins containing N-linked oligosaccharides. DISCUSSION Abnormalities in glycoconjugate metabolism have been reported to be due to incorrect carbohydrate transfer and processing, improper protein structure, or lack of gene expression (see review (19)). In NCL, abnormalities in glycoconjugate metabolism particularly in the carbohydrate portion have been reported in the brain (9,11-13) skin (lo), and cultured fibroblasts (2,8) and the defects usually involve the high mannose type of N-linked oligosaccharide. In studies of the accumulated oligosaccharyldiphosphodolichols of NCL, the oligosaccharides appear to be metabolites of the normal glycosylation intermediate Glc3MangGlcNA@ (20-22), w h ic h would result from a block in the transfer to a protein acceptor or a deficiency in processing enzymes. In this study both carbohydrate and protein of the M, 120-140 kDa glycoproteins were diminished and either or both may be defective. Since there are several reports of the carbohydrate defects in NCL, two instances of other disorders may be cited in which the incorrect structure of N-linked carbohydrate leads to degradation of the glycoproteins. In a disorder of sucrase-isomaltase, an enzyme of the intestinal brush border, the high mannose precursor is transferred properly to the protein, but processing to the mature complex oligosaccharide is defective leading to degradation of the glycoprotein (23). In the familial form of hypercholesterolemia, one defect of the LDL receptor is an incorrect processing of the precursor oligosaccharide leading to degradation of the glycoprotein (24). The three childhood forms of NCL are classified clinically by age of onset and course of the disease and, although the forms share characteristics of vision loss, seizures, myoclonus, and dementia, it is not clear whether these are separate diseases since few biochemical studies have compared the childhood forms. The juvenile and infantile forms have autosomal inheritance. Recent genetic work has assigned the infantile form in Finnish families to Chromosome 1 (25) and the juvenile form in western European families to Chromosome 16 (26). In a study of oligosaccharides derived from dolichol intermediates, the juvenile form had smaller average size and the infantile form larger size (20-22). In the present study of labeled glycoconjugates of fibroblasts, the infantile form showed a 75%
GLYCOPROTEIN
METABOLISM
IN NCL
141
reduction of M, 120-140 kDa glycoproteins while the juvenile form did not, suggesting that the two forms of NCL differ qualitatively at least in these components. These differences may be useful in the diagnosis of these two forms of NCL. ACKNOWLEDGMENTS This work was supported in part by funds from NIH Grant NS 23717, BRSG 2-SO7 RR05838-11, and by funds from the NYS Office of Mental Retardation and Developmental Disabilities.
REFERENCES 1. Zeman W. The neuronal ceroid-lipofuscinoses. Prog NeuropathoZ3:203-223, 1976. 2. Wisniewski KE, Rapin I, Heaney-Kieras J. Clinico-pathological variability in the childhood neuronal ceroid-lipofuscinoses and new observations on glycoprotein abnormalities. Am J Med Genet Suppl5:27-46, 1988. 3. Siakotos AN, Bray R, Dratz E, Van Kuijk F, Sevanian A, Koppang N. 4-Hydroxynonenal: A specific indicator for canine neuronal-retinal ceroidosis. Am J Med Genet Suppl5:171-181, 1988. 4. Pullarkat RK, Kim KS, Sklower SL, Pate1 VK. Ohgosaccharyl diphosphodolichols in the ceroidhpofuscinoses. Am J Med Genet Suppl5:243-251, 1988. 5. Hall NA, Patrick AD. Accumulation of dolichol-linked oligsaccharides in ceroid-lipofuscinosis (Batten Disease). Am J Med Genet Suppl5:221-232, 1988. 6. Palmer DN, Martinus RD, Barns G, Reeves RD, Jolly RD. Ovine ceroid-lipofuscinosis 1:Lipopigment composition is indicative of a lysosomal proteinosis. Am J Med Genet SuppZ5:141158, 1988. 7. Dawson G, Glaser PT. Abnormal Cathepsin B activity in Batten Disease. Am J Med Genet Suppl 5:209-220, 8.
9. 10. 11.
12. 13. 14. 15. 16.
1988.
Dawson G. Approaches to the detection of neuronal ceroid lipofuscinosis in cultured skin tibroblasts. In Ceroid-Lipofuscinosis (Batten’s Disease) (Amrstrong D, Koppang N, Rider JA, Eds.). Amsterdam: Elsevier, 1982, pp 229-240. Krusius T, Viitala J, Palo J, Maury CP. Enrichment of high mannose-type glycans in nervous tissue glycoproteins in neuronal ceroid-lipofuscinosis. J Neural Sci 72:1-10, 1986. Wisniewski KE, Szumanska G. The ultrastructural observation and histochemical localization of some glycoconjugates in neuronal ceroid-lipofuscinosis. 10th Intl Congr Neuropathol, Stockholm, Sweden (No.799.2) p 396, 1986. Wisniewski KE, Maslinska D, Kitaguchi T. High level mannose type of glycopeptides in brains of human neuronal ceroid-lipofuscinosis (HNCL) and canine experimental model (CNCL). J Neuropath Exp Neural 47~327, 1988. Elleder M. Lectin histochemical study of lipopigments with special regard to neuronal ceroidlipofuscinosis. Results with concanavalin A. Histochemistry 93197-205, 1989. Wisniewski KE, Maslinska D. Lectin histochemistry in brains with juvenile form of neuronal ceroid-lipofuscinosis (Batten Disease). Acta Neuropathol80:274-79, 1990. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the fohn-phenol reagent. J Biol Chem 193~265-275, 1951. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685, 1970. Tarentino AL, Plummer TH Jr. Peptide-N“-(N-acetyl-P-glucosaminyl) asparagine amidase and endo-/3-N-acetylglucosaminidase from Flavobacterium meningosepticum. Methods Enzymol D&770-778,
1987.
Hirani S, Bernasconi RJ, Rasmussen JR. Use of N-Glycanase to release asparagine-linked oligosaccharides for structural analysis. Anal Biochem 162~485-492, 1987. 18. Freeze HH, Varki A. Endo-glycosidase F and peptide N- glycosidase F release the great majority of total cellular N-linked oligosaccharides: Use in demonstrating that sulfated N-linked oligosaccharides are frequently found in cultured cells. Biochem Siophys Res Commun 140~967-973, 1986. 17.
142
HEANEY-KIERAS,
KIERAS,
AND WISNIEWSKI
19. Rademacher TW, Parekh RB, Dwek RA. Glycobiology. Annu Rev Biochem 57~785-838, 1988. 20. Hall NA, Patrick AD. Analysis of dolichol-linked oligosaccharides in brains from patients with Batten’s disease. B&hem Sot Truns 16:1031-1032, 1988. 21. Hall NA, Patrick AD. Identification of intact dolichol-linked oligosaccharides in the brains of patients with ceroid lipofuscinosis (Batten’s disease). J Inher Metub Dis Suppl2:379-382, 1989. 22. Hall NA, Haltia M, Patrick AD. High mannose dolichol-linked oligosaccharides in infantile ceroid lipofuscinosis. Biochem Sot Trans 17~1032-1033, 1989. 23. Lloyd ML, Olsen WA. A study of the molecular pathology of sucrase-isomaltase deficiency. N Engl J Med 316~438-442, 1987. 24. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science 23234-47, 1986. 25. Jarvell I, Mbnpil J, Haataja L, Santovouri P, Aula P, Peltonen L. Assignment of infantile form of neuronal ceroid-lipofuscinosis (INCL) to the short arm of chromosome 1. Am J Med Genet 47A185, 1990. 26. Eiberg H, Gardiner RM, Mohr J. Batten disease (Spielmeyer-Sjogren disease) and haptoglobins (HP): Indication of linkage and assignment to chr. 16. Clin Genet 36~217-218, 1989.
